A human-specific enhancer fine-tunes radial glia potency and corticogenesis

Humans evolved an extraordinarily expanded and complex cerebral cortex, associated with developmental and gene regulatory modifications1-3. Human accelerated regions (HARs) are highly conserved DNA sequences with human-specific nucleotide substitutions. Although there are thousands of annotated HARs, their functional contribution to species-specific cortical development is largely unknown4,5. HARE5 is a HAR transcriptional enhancer of the WNT signaling receptor Frizzled8 (FZD8) active during brain development6. Here, using genome-edited mouse and primate models, we demonstrate that human (Hs) HARE5 fine-tunes cortical development and connectivity by controlling the proliferative and neurogenic capacity of neural progenitor cells (NPCs). Hs-HARE5 knock-in mice have significantly enlarged neocortices, which contain more excitatory neurons. By measuring neural dynamics in vivo we show these anatomical features result in increased functional independence between cortical regions. To understand the underlying developmental mechanisms, we assess progenitor fate using fixed and live imaging, lineage analysis, and single-cell RNA sequencing. We discover Hs-HARE5 modifies radial glial progenitor behavior, with increased self-renewal at early developmental stages followed by expanded neurogenic potential later. We use genome-edited human and chimpanzee (Pt) NPCs and cortical organoids to assess the relative enhancer activity and function of Hs-HARE5 and Pt-HARE5. Using these orthogonal strategies we show four human-specific variants in HARE5 drive increased enhancer activity which promotes progenitor proliferation. Finally, we show that Hs-HARE5 promotes progenitor proliferation by increasing canonical WNT signaling. These findings illustrate how small changes in regulatory DNA can directly impact critical signaling pathways to modulate brain development. Our study uncovers new functions for HARs as key regulatory elements crucial for the expansion and complexity of the human cerebral cortex.